The neuronal circuitry underlying sleep-wakefulness is beginning to be understood. Changes in the release of neurotransmitters across sleep-wakefulness are well documented. The intracellular events associated with the activity of these neurons across sleep-wakefulness, however, are not clear. The available evidence suggests that the transcriptional activation of genes occurs during sleep, indicating that changes in protein and mRNA expression are likely to occur during sleep as well as waking states. The function of sleep is not known. One hypothesis is that sleep serves to replenish biomolecules depleted during waking. If sleep serves such a function, The intracellular environment of brain centers involved in sleep- wakefulness will support biosynthesis. The main hypothesis of this proposal is that changes in the expression of key enzymes associated with neurotransmitter synthesis occur sleep- wakefulness to maintain neurotransmitter stores. To test this hypothesis, the expression of choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine will be examined. Specific aim 1 will test the hypothesis that chat mRNA is increased during sleep. In these experiments, mRNA levels will be examined during waking, non-REM and REM sleep states that occur across a 24-hour period. Specific aim 2 tests the hypothesis that protein activity and/or protein levels vary across individual sleep bouts over a 24-hour period. Specific aims 3 and 4 will test the hypothesis that sleep loss effects mRNA expression (Specific aim 3) and protein expression (Specific aim 4). In these experiments, the reversibility of the effects of sleep loss will also be monitored during the recovery sleep period. Sleep disorders affect approximately 70 million Americans with associated costs estimated at billions of dollars in lost revenue per year. By identifying how sleep might replenish key enzymes in neurotransmitter synthesis, we will advance our knowledge of sleep. The experiments proposed are the first steps in the identification of cellular mechanisms effecting acetylcholine synthesis across sleep-wakefulness.